Enzyme-producing strain of Bacillus bacteria

ABSTRACT

This invention presents a newly discovered, novel strain of Bacillus bacteria that produces lipase enzymes for the degradation of oleaginous materials such as fats, greases and cooking oils, protease enzymes to degrade proteins and amylases to break down starch. This novel strain and the enzymes produced thereby have a number of applications, including wastewater treatments, agricultural uses, laundry and dish detergents, drain cleaners and spot removers, among others.

FIELD OF THE INVENTION

This invention consists of a newly discovered, novel strain of Bacillusbacteria which produce multiple useful enzymes. The novel bacterium andthe enzymes it produces have a number of applications, includingwastewater treatment, agricultural uses, laundry and dish detergents,drain cleaners and spot removers, among others. In particular, theBacillus strain of the present invention produces lipase enzymes for thedegradation of oleaginous materials such as fats, greases and cookingoils, as well as protease enzymes to degrade proteins and amylases tobreak down starch.

BACKGROUND OF THE INVENTION

The use of bioaugmentation, i.e., the addition of non-indigenousbacteria to a wastewater to effect more complete treatment andpurification of the water, is becoming more and more necessary asmunicipal sewage plants age and demands brought on by increasing loadsrequire improvements in efficiency. Bioaugmentation, also calledbacterial augmentation, can be beneficial in a number of applications.For example, bacterial augmentation can improve the quality andefficiency of treatment of municipal wastewater, food processingwastewater, and residential wastewater, especially in on-site disposalsystems such as septic tanks and cesspools, pretreatment of wastewaterthat might contain high levels of oleaginous matter, and treatment ofpipes, traps, plumbing systems and grease traps.

Enzyme-producing bacteria also have agriculture uses as silage inoculantproducts to improve efficiency and production of livestock that consumethe treated silage. Certain bacteria, called Direct Fed Microbials, mayalso be added to livestock feeds to increase food digestion andutilization. Currently both Bacillus subtilis and Bacillus pumilus havewere found by the Food and Drug Administration to present no safetyconcerns when used in direct-fed microbial products, as published by theAssociation of American Feed Control Officials (AAFCO). 1999 OfficialPublication of the Association of American Feed Control Officials, p.221. In addition, advances in bioengineering have demonstrated that agenetic characteristic from one species, including bacteria, can beinserted into the DNA of another species. One important example is theinsertion of the gene responsible for the production of an insecticidalprotein from Bacillus thuringiensis into the DNA of corn seeds. Theresultant corn plant is able to produce a protein which kills insectpests such as the corn rootworm, thereby producing healthier corn plantsand improved per-acre yields.

SUMMARY OF THE INVENTION

The object of this invention therefore is to provide a novel strain ofbacteria which produces specific types of useful enzymes.

It is a further object of the present invention to provide a novelstrain of bacteria that produces lipase enzymes to degrade fat,proteases to degrade proteins, and amylases to degrade starch.

It is another object of the present invention to provide a novelmultiple-enzyme-producing bacterial strain that is useful for wastewaterapplications to enhance degradation of the organic components of thewastewater and therefore improve the quality and efficiency of the watertreatment process.

It is yet a further object of the present invention to provide a novelmultiple-enzyme-producing strain of bacteria for use in agriculturalapplications, including as Direct Fed Microbials in livestock feedformulations, as silage innoculants and to treat livestock manure.

It is a still further object to provide novel bacteria and/or enzymesproduced from such bacteria for use as detergents, and to provideenzymes to use as stain removers or in food processing applications.

The present invention provides a novel stain of Bacillus bacteria thatproduces lipases to degrade fat, proteases to degrade proteins, andamylases to degrade starch. The novel strain disclosed and claimed inthe present application is one of eight novel strains invented by thepresent inventors. For completeness of description, the specificationwill describe all eight strains, but only the Bacillus strain designatedas ATCC 202137 is claimed in the present application. The eight novelstrains of Bacillus bacteria have all been deposited with the AmericanType Culture Collection ("ATCC"), 10801 University Blvd., Manassas, Va.,20110-2209, U.S.A., on Jun. 5, 1998, under the rules of the BudapestTreaty and are designated as follows:

    ______________________________________                                        Species          Strain     ATCC Number                                       ______________________________________                                        Bacillus pumilus RLM-002    ATCC 202136                                         Bacillus subtilis RLM-007Aa ATCC 202138                                       Bacillus amyloliquefaciens RLM-007Ab ATCC 202134                              Bacillus macerans RLM-007C ATCC 202132                                        Bacillus subtilis RLM-011 ATCC 202137                                         Bacillus subtilis RLM-012A ATCC 202139                                        Bacillus amyloliquefaciens RLM-012B ATCC 202133                               Bacillus macerans RLM-013C ATCC 202135                                      ______________________________________                                    

The species of each of these Bacillus bacterial strains were determinedusing the Fatty Acid Methyl Ester (FAME) Analysis method performed byMicrocheck, Inc., of Northfields, Vermont using Trypticase Soy BrothAgar ("TSBA") version 3.9 at 28.0 C of Microcheck's database.

The eight novel Bacillus strains have a variety of potential uses,including treatment of wastewater, such as municipal, food processing,and residential wastewater; pretreatment of wastewater that mightcontain high levels of oleaginous matter; treatment of on site sewagedisposal systems, such as septic tanks and cesspools; agricultural uses,including livestock manure waste management or as Direct Fed Microbialsfor use in livestock feeds to increase food digestion and utilization;and for treatment of pipes, traps, plumbing systems and grease traps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 charts the production of lipase enzymes at various points withina temperature range by Bacillus amyloliquefaciens strains RLM-007Ab,ATCC 202134 and RLM-012B, ATCC 202133.

FIG. 2 charts the production of lipase enzymes at various points withina temperature range by Bacillus macerans RLM-013C, ATCC 202135 andRLM-007C, ATCC 202132, and Bacillus pumilus RLM-002, ATCC 202136.

FIG. 3 charts the production of lipase enzymes at various points withina temperature range by Bacillus subtilis RLM-011, ATCC 202137, RLM-012A,ATCC 202139, and RLM-007Aa, ATCC 202138.

FIG. 4 shows the data generated as a result of the Fatty Acid MethylEster (FAME) Analysis method performed by Microcheck, Inc., ofNorthfields, Vt. using TSBA version 3.9 at 28.0 C of Microcheck'sdatabase, which was used to determine the species of Bacillus subtilisATCC 202137.

DETAILED DESCRIPTION OF THE INVENTION

The eight newly discovered strains of Bacillus bacteria were isolatedfrom soil using standard microbiological methods. These strains wereisolated using a method which was adapted from Bergey's Manual ofSystematic Bacteriology, Vol. 2, pages 1104-1139. The procedure used wasselective for the genus Bacillus because of this genus' ability tosurvive the pasteurization process by producing spores that areresistant to temperatures reached during the pasteurization process. Inthe procedure, approximately one gram of soil from the vicinity ofSeattle, Wash., was added to 300 ml of Tryptic Soy Broth (Difco). Thismixture was then pasteurized by heating to 70 C for 10 minutes. Theflask was then incubated for 48 hours at 30 C. The culture was struckonto (TSA) Tryptic Soy Agar (Difco) plates at the beginning of theincubation period, following 24 hours of incubation, and at the end ofthe 48 hours of incubation. These plates were then incubated aerobicallyat 30 C and examined for their bacterial growth. Bacterial colonies werepicked and serially transferred onto TSA plates approximately every 24to 48 hours until stable bacterial colonies could be isolated.

Taxonomic determination was accomplished via colonial morphology,cellular morphology, Gram stain, biochemical tests, and Fatty AcidMethyl Ester (FAME) Analysis performed by Microcheck, Inc., ofNorthfields, Vt. using TSBA version 3.9 at 28.0 C of Microcheck'sdatabase.

All eight strains of the of Bacillus bacteria of the present inventionare Gram positive rod shaped bacteria that grow under aerobicconditions. As is typical of the genus, they bear spores that germinateto vegetative reproducing cells.

The newly discovered Bacillus strains of the present invention arepresented in Table I. The Roebic Laboratories Microbe (RLM) number isgiven with the species identification as provided by FAME Analysismethod performed by Microcheck, Inc., of Northfields; Vt. using TSBAversion 3.9 at 28.0 C of Microcheck's database.

Also, the right column provides the similarity indices/standarddeviations (SI/SD).

                  TABLE I                                                         ______________________________________                                        Reference                                                                              Species Identification SI/SD                                         ______________________________________                                        RLM-002  Bacillus pumilus GC subgroup B                                                                       0.628                                           RLM-007Aa Bacillus subtilis 0.475                                             RLM-007Ab Bacillus amyloliquefaciens 0.608                                    RLM-007C Paenibacillus macerans GC subgroup A 0.729                           RLM-011 Bacillus subtilis 0.519                                               RLM-012A Bacillus subtilis 0.435                                              RLM-012B Bacillus amyloliquefaciens 0.593                                     RLM-013C Paenibacillus macerans GC subgroup A 0.415                         ______________________________________                                    

In order to more fully describe each Bacillus strain, additionalbiochemical tests were performed to demonstrate the ability to utilizevarious carbohydrates as nutrient sources, and to use urea and citrateas sources of assimilable carbon. The results are presented in Table II.

                                      TABLE II                                    __________________________________________________________________________    CARBOHYDRATE UTILIZATION                                                                Strain RLM-                                                         Carbohydrates Tested                                                                    002                                                                              007Aa                                                                             007Ab                                                                             007C                                                                             011                                                                              012A                                                                              012B                                                                              013C                                       __________________________________________________________________________    Adonitol  -  -   -   -  -  -   -   -                                            Arabinose - - - - + - - -                                                     Dulcitol - - - - - - - -                                                      Glucose - + + + + + + +                                                       Lactose - - - - - - - -                                                       Maltose - - - - - - - -                                                       Mannitol - - + - + - + +                                                      Sorbitol - - - + + - - -                                                      Sucrose + + + + - - + -                                                       Xylose - - - - - - - -                                                        Carbon Sources Tested                                                         Urea - + + + + + + +                                                          Citrate - - - + + - + +                                                     __________________________________________________________________________

Results from either Becton Dickenson Enterotube II or Oxi/Ferm Tube IIat 48 hours at 34 C. These tests were conducted following themanufacturer's instructions and were inoculated with 24 hour Tryptic SoyAgar ("TSA") colonies.

All eight Bacillus strains except Strain RLM-002 (ATCC 202136, were ableto reduce nitrate to nitrite, which is a useful characteristic for thetreatment of wastewater where dissolved oxygen levels are low, butnitrate levels are high. This would include on-site sewage disposalsystems such as septic tanks and cesspools.

To further identify the specific capabilities of each Bacillus strain,tests were conducted using Tryptic Soy Broth (Difco) ("TSB") todetermine the minimum and maximum temperature at which each would grow.The test was conducted using test tubes of TSB incubated at theindicated temperature for 14 days. The results are presented in TableIII.

                                      TABLE III                                   __________________________________________________________________________    GROWTH TEMPERATURE RANGE                                                      Strain                  10 C.                                                                            15 C.                                                                            40 C.                                                                            45 C.                                                                            50 C.                                     __________________________________________________________________________    RLM-002                                                                              B. pumilus                                                                              ATCC 202136                                                                          +  +  +  +  +                                           RLM-007Aa B. subtilis ATCC 202138 + + + + -                                   RLM-007Ab B. amyloliquefaciens ATCC - + + + -                                 202134                                                                        RLM-007C B. macerans ATCC 202132 - + + + -                                    RLM-011 B. subtilis ATCC 202137 + + + + +                                     RLM-012A B. subtilis ATCC 202139 - + + + -                                    RLM-012B B. amyloliquefaciens ATCC - + + + -                                  202133                                                                        RLM-013C B. macerans ATCC 202135 - + + + -                                  __________________________________________________________________________

The fact that all eight Bacillus strains were able to grow at 15 C isparticularly useful for wastewater treatment and agriculturalapplications. Also, two strains of Bacillus subtilis, RLM-007Aa andRLM-01 1, and Bacillus pumilus RLM-002 were able to grow at 10 C. Thelatter strain, along with Bacillus subtilis RLM-011, was able to grow at50 C as well. These two organisms exhibited the widest temperature rangewithin the group of eight strains. Growth over such a wide temperaturerange significantly expands the areas of usefulness of these novelbacteria to include many applications in the environment.

Table IV identifies the useful enzymes produced by the newly discoveredeight Bacillus strains

                                      TABLE IV                                    __________________________________________________________________________    USEFUL ENZYMES PRODUCED                                                                                       Proteases                                     Strain                 Amylase                                                                            Lipase                                                                            Gelatin                                                                           Casein                                    __________________________________________________________________________    RLM-002                                                                             B. pumilus                                                                              ATCC 202136                                                                          -    +   +   +                                           RLM-007Aa B. subtilis ATCC 202138 + + + +                                     RLM-007Ab B. amyloliquefaciens ATCC + + + +                                   202134                                                                        RLM-007C B. macerans ATCC 202132 - + + -                                      RLM-011 B. subtilis ATCC 202137 + + + +                                       RLM-012A B. subtilis ATCC 202139 + + + +                                      RLM-012B B. amyloliquefaciens ATCC + + + +                                    202133                                                                        RLM-013C B. macerans ATCC 202135 + + + +                                    __________________________________________________________________________

EXAMPLE 1

The production of amylase enzymes by bacteria is a useful characteristicwith many applications including wastewater treatment and foodprocessing methods. The production of amylase enzymes by the novelstrains of the present invention was determined through the use of BactoStarch Agar (Difco). Simple streaks were used as well as disk zonehydrolysis. Bacto Starch Agar consists of Bacto Beef Extract (3 g/l),soluble starch (10 g/l), and Bacto Agar (12 g/l). To prepare starch agarplates, 25 g/l of Bacto Starch Agar was suspended in de-ionized water.The water was heated so that the starch dissolved completely. Theaqueous starch solution was then sterilized for 15 minutes at 120 C andallowed to cool. The media was then poured into plates and allowed tosolidify and dry overnight.

For the streak test, a sterile platinum inoculating needle was used tostreak a pure strain onto an individual starch agar plate. The plateswere then incubated at 30 C. At 24 hours, the plates were flooded withGram's Iodine solution. This procedure was repeated at 48 hours. Apositive result was indicated by a yellow zone surrounding the bacterialgrowth. If the starch had not been hydrolyzed, then the agar wouldappear as a very dark purple.

For the disk zone hydrolysis test, sterile paper disks were applied tothe surface of dry starch agar plates and were then soaked with a24-hour pure culture. These plates were then incubated for 24 hours andthen flooded with Gram's Iodine solution. This test was also repeatedfollowing 48-hour incubation. A positive result was indicated by ayellow zone surrounding the disk.

Table V is a summary of amylase production by the bacteria strains ofthe present invention.

                  TABLE V                                                         ______________________________________                                        RESULTS OF AMYLASE TESTING                                                      Streak Test at 48 Hours                                                                           Disk Zone Hydrolysis Test at 48 Hours                   Strain   +/-      Strain     +/-    Zone (mm)*                                ______________________________________                                        RLM-002  -        RLM-002    -      0                                           RLM-007Aa + RLM-007Aa + 2                                                     RLM-007Ab + RLM-007Ab + 2                                                     RLM-007C - RLM-007C - 0                                                       RLM-011 + RLM-011 +   2.5                                                     RLM-012A + RLM-012A + 1                                                       RLM-012B + RLM-012B + 1                                                       RLM-013C + RLM-013C +   1.5                                                 ______________________________________                                         *Measurement was made from the edge of the colony or paper disk to the        edge of the zone of hydrolysis.                                          

The data indicate that all strains except RLM-002 and RLM-007C producedamylase enzyme. Bacillus subtilis RLM-011 produced the largest zones andpresumably the most enzyme.

EXAMPLE 2

The production by bacteria of proteases, or protein degrading enzymes,is also a valuable trait with many applications. The commercial use ofsuch bacteria in wastewater treatment, food processing and laundrydetergents presents a substantial commercial market for these bacteriaand the enzymes they produce.

The eight novel discovered Bacillus strains were tested with twoproteinaceous materials, casein and gelatin, and all strains displayedvarying abilities to degrade these proteins. The production of proteaseenzymes was determined through the analysis of casein hydrolysis andnutrient gelatin liquefaction. Bacto Nutrient Gelatin (Difco) consistsof Bacto Beef Extract (3 g/l), Bacto Peptone (5 g/l), and Bacto Gelatin(120 g/l). To prepare Nutrient Gelatin tubes, 128 grams of BactoNutrient Gelatin was suspended in 1 liter of de-ionized water. The waterwas heated so that the gelatin dissolved completely and the aqueousmixture was distributed by 15 milliliter aliquots into test tubes. Thetubes were sterilized in the autoclave for 15 minutes at 121 C, and werethen allow to cool and solidify. An isolated colony from a TSA plate wastouched with a sterile inoculating needle and the needle was stabbeddown through the Nutrient Gelatin Agar. The tubes were then incubatedfor 4 days at 37 C. After the incubation period, the tubes were removedand allowed to cool. A positive result is indicated if any of thegelatin remains liquefied and does not re-solidify.

Casein hydrolysis was tested for both through Bushnell-Haas-Casein("BHC") plates and then through disk zone analysis on TSA-Casein plates.

Bushnell Haas media is available for purchase pre-made (from DifcoLabratories or a local distributor). It consists of magnesium sulfate(0.2 g/l), calcium chloride (0.02 g/l), monopotassium phosphate (1 g/l),dipotassium phosphate (1 g/l), ammonium nitrate (1 g/l), and ferricchloride (0.05 g/l). Casein, technical grade, and Agar are bothavailable from Difco.

To prepare the Bushnell-Haas-Casein agar plates, 3.25 g/l Bushnell Haasmedium was dissolved in I liter de-ionized water. To the aqueousmixture, 20 g/l Casein and 20 g/l agar were added sequentially. Themixture was heated so all solutes dissolved completely and was thensterilized at 120 C for 15 minutes. The agar was allowed to cool, pouredinto plates and allowed to solidify. The plates so prepared wereinoculated by touching a sterile inoculating needle to an isolatedcolony on a TSA plate, and streaking the needle once across the surfaceof the BHC agar plate. The streaked plates were incubated for 48 hoursat 30 C, and then held at room temperature for two additional days. Apositive result was indicated by a clearing of the casein.

TSA-Casein Agar plates were prepared using Difco TSA to which 20 g/lCasein, technical grade, was added. TSA consists of Bacto Tryptone (15g/l), Bacto Soytone (5 g/l), Sodium Chloride (5 g/l), and Bacto Agar (15g/l). To prepare TSA-Casein plates, 40 g/l TSA was suspended in 1 literof de-ionized water, and twenty grams of Casein was then added. Theaqueous mixture was heated so that the solutes dissolve completely, andthen sterilized at 120 C for 15 minutes. The mixture was allowed tocool, was poured into plates, and was allowed to solidify and dryovernight. In the center of the each agar plate, a paper disk was placedwhich was then soaked with a few drops of a 24-hour TSB pure culture ofthe test organism. The plates were then incubated and examinedperiodically. A positive result showed clearing of the casein fromaround the disk within 48 hours.

The following is a summary of protease production as determined bycasein hydrolysis (Table VI) and gelatin liquefaction (Table VII).

                  TABLE VI                                                        ______________________________________                                        RESULTS OF CASEIN HYDROLYSIS TESTING                                                                TSA-Casein Disk Zone Hydrolysis Test                      BH-Casein Streak Test  at 48 Hours                                          Strain   +/-      Strain     +/-    Zone (mm)*                                ______________________________________                                        RLM-002  -        RLM-002    +      2                                           RLM-007Aa + RLM-007Aa + 1-2                                                   RLM-007Ab - RLM-007Ab + 1-2                                                   RLM-007C - RLM-007C - 0                                                       RLM-011 + RLM-011 + 3-5                                                       RLM-012A + RLM-012A + 3                                                       RLM-012B + RLM-012B + 3-4                                                     RLM-013C + RLM-013C + 3-4                                                   ______________________________________                                         *Measurement was made from the edge of the colony or paper disk to the        edge of the zone of hydrolysis.                                          

                  TABLE VII                                                       ______________________________________                                        RESULTS OF NUTRIENT GELATIN TESTING                                                              Day 2    Day 4                                               Strain (48 Hrs.) (96 Hrs.)                                                  ______________________________________                                        RLM-002        -        +                                                       RLM-007Aa - +                                                                 RLM-007Ab - +                                                                 RLM-007C - +                                                                  RLM-011 + +                                                                   RLM-012A - +                                                                  RLM-012B + +                                                                  RLM-013C - +                                                                ______________________________________                                    

EXAMPLE 3

The production of lipase, or fat splitting enzyme(s), by the eight novelBacillus strains of the present invention is an important characteristicthat has significant commercial applications.

All enzymes perform under a range of conditions, most having a maximumlevel of activity at a given, sometimes narrow, temperature range.Lipolytic activity therefore was tested at several temperatures todemonstrate the active range of the lipase produced as well as thetemperature at which it has optimal activity. The test for lipaseproduction utilized Bacto Spirit Blue Agar with Bacto Lipase Reagent(Difco), which is a widely-accepted medium for detecting lipolyticmicroorganisms. According to its label, the formula for Spirit Blue Agarcomprises Bacto Tryptone (10 g/l), Bacto Yeast Extract (5 g/l), BactoAgar (20 g/l), and Spirit Blue (0.15 g/l). This medium can be purchasedalready prepared (from Difco Laboratories or a local distributor), ormay be prepared as follows: Thirty-five grams of dried medium issuspended in 1 liter of distilled or de-ionized water and the aqueousmixture is heated to boiling so that the solutes are dissolvedcompletely. The solution is then sterilized in an autoclave for 15minutes at 15 pounds of pressure and 121 C. It is cooled to 50-55 C, and30 ml of Bacto Lipase Reagent is added aseptically and mixed thoroughly.The desired amount is poured into a 120 millimeter (Falcon 1020)disposable petri dish. The inventors are not aware of the formulationfor the lipase reagent, but the reagent can be purchased from Difco.

Spirit Blue Agar with Lipase Reagent plates (SBLP) were held at roomtemperature overnight in order to both solidify and to dry the surfaceof the agar. Lipase enzymatic activity was tested by simple bacterialstreaking, incubation, and quantitative measuring of the zones ofclearing. To streak a plate, a sterile platinum inoculating loop wastouched to an isolated colony grown on TSA for a minimum of 48 hours.The loop was then carefully struck twice back and forth across thesurface of an SBLP. Following incubation, this procedure yields acontinuous and luxuriant growth of bacteria which bifurcates the SBLPplate and allows for the activity of lipase enzymes to be observed overa wide time frame. Plates initially were incubated at about 30 C for 24hours, zones of clearing were measured, and the plates were then held atroom temperature for approximately 7 days during which time additionalmeasurements and observations were made. In evaluating lipolyticactivity, simple streaks were used as well as measurements of zones ofclearing.

This streak procedure was carried out again at various incubationtemperatures which were kept constant throughout the 7 day test in orderto elucidate the particular strain's enzymatic activity curves over awider temperature range. Each strain's lipolytic activity was tested at10 C, 15 C, 20 C, and 30 C. A positive lipolytic result was indicated bya clearing in the agar medium's opacity immediately surrounding thebacterial growth. The larger this zone of clearing is, the greater thelipase activity for the bacterial strain. Color change in the medium isnot fully indicative of lipolytic activity.

The results of this testing are presented on Table VIII.

                  TABLE VIII                                                      ______________________________________                                        RESULTS OF LIPASE TESTING: ZONES OF CLEARING (mm)                               ON DAY 7 AT GIVEN TEMPERATURE RANGES (Streak Tests)                                     Incubation Temperature                                            Strain      10 C.  15 C.      20 C. 30 C.                                     ______________________________________                                        RLM-002     1      3          4     5                                           RLM-007Aa 1 2-3 2 2-3                                                         RLM-007Ab 1 2 6 3-4                                                           RLM-007C 1 3   6.5 6                                                          RLM-011 1 3 4 7                                                               RLM-012A 1 3 5 7                                                              RLM-012B <1   3 3.5-4   2                                                     RLM-013C   1.5 2-3 2-3 5-7                                                  ______________________________________                                    

The data in Table VIII, which are also represented in graph form. inFIGS. 1, 2, and 3, clearly demonstrates the production of lipase enzymescapable of splitting fats, grease, oils or other oleaginous materials(lipid hydrolysis). The figures compare the relationship betweentemperature and lipase enzyme production among the Bacillus strainsgrouped by species.

While it is generally recognized that bacterial metabolism increaseswith temperature as it approaches an optimum, FIG. 1 clearlydemonstrates that the two Bacillus amyloliquefaciens strains RLM-007Ab,ATCC 202134 and RLM-012B, ATCC 202133 produced more lipase activity at20 C than at 30 C. This characteristic easily positions these strains asthe bacteria of choice for use in lower temperature environments such aswastewater treatment or food processing waste treatment in holding tanksor lagoons. It is expected that this characteristic would provide to atreatment product containing one or both of these microbes an advantageover a competitive product that did not perform as well at thattemperature.

The bacteria represented on FIG. 2 demonstrate a different pattern oflipase activity. Bacillus macerans RLM-013C, ATCC 202135 producedsignificantly more lipolytic activity at 30 C than at 20 C. The otherstrain of Bacillus macerans, RLM-007C, ATCC 202132, behaved quitedifferently, exhibiting a dramatic increase in activity from 15 C to itspeak at 20 C, which dropped off slightly as the temperature increased to30 C. The Bacillus pumilus RLM-002, ATCC 202136 generally demonstratedincreased activity with increasing temperature to a maximum of lipolyticactivity at 30 C.

The three strains of Bacillus subtilis represented in FIG. 3 displayed athird pattern of activity. Two strains, RLM-011, ATCC 202137, andRLM-012A, ATCC 202139, both produced more lipolytic activity as thetemperature increased, but strain RLM-007Aa, ATCC 202138, showed no suchincrease. Its lipase activity was considerably lower overall, andreached its peak at 15 C.

These varying patterns of lipolytic activity among the Bacillus strainsprovide the basis for combinations of two or more strains for variousapplications in which temperature is a factor. Such information is alsouseful for the mass production of the strains in "fermentors," which arelarge culture vessels for preparing or "fermenting" batches of bacteria,yeasts, molds, etc. Regardless of whether the bacteria are fermented tobe sold as whole cultures, spore concentrates, or if the enzymes were tobe harvested and purified for specific market uses, temperaturedifferences play a key role in the selection of strains for particularapplications.

All eight Bacillus strains showed evidence of lipolytic activity at 10 Cin this examination, while only three strains actually grew at 10 C inthe temperature study (Table III). The difference in methods and growthmedia are factors in this apparent difference, but it is also morelikely that, although some metabolic activity occurred, such as thelimited production of extracellular lipase enzyme, overall activity at10 C was insufficient for cellular growth and reproduction such as thatwhich would be required to produce positive results in the temperaturegrowth study. These data are encouraging, however, in that it could bepossible with certain nutrient selection, or increased oxygen, toculture these organisms more robustly at 10 C in a lipid environment.

Myriad uses are envisioned for the products of the present invention.The claimed bacteria and/or the enzymes produced thereby can be used ina number of wastewater treatment or plumbing system applications,including treatment of municipal or residential wastewater, treatment ofon site sewage disposal systems, such as septic systems, and cesspools,pretreatment and/or treatment of food processing wastewater, and asdrain openers or cleaners for waste lines, sinks, tubs or other plumbingsystems. The claimed bacteria or enzymes may also be used inagricultural applications, such as for Direct Fed Microbials, which canbe added to livestock feed to increase food digestion and utilization,as silage innoculants and to treat livestock manure. The claimedbacteria or enzymes may be useful as detergents in a variety ofapplications, and the enzymes could be used in spot removers or for foodprocessing applications.

The bacteria of the present invention can also provide an original genepool for recombinant bacteria applications and further recombinant DNAwork. It is probable, given the current knowledge and sophistication ofrecombinant genetics, that the gene(s) controlling lipase productionfrom one or all of these bacteria could be inserted into the DNA ofother species to deliver the capability to the receiving species. If thereceiving species is a food for humans or livestock, it could become thecarrier for the characteristic to enhance food processing andutilization.

The lipase enzymes recovered from the fermentation of such Bacillusbacteria are expected to have a variety of uses, including in foodpreparation and processing, or in detergents or cleaners to degrade fatsand greases on clothing, linens and on surfaces in kitchens and otherfood preparation areas, and in sinks, drain lines and grease traps.

Although the invention has been described in detail and with referencesto applications in several areas known to utilize beneficial bacteriafor process improvement and greater efficiencies, it is acknowledgedthat it will be apparent to one skilled in the art that modifications,changes, and formulations with other biological or chemical ingredientscan be made without departing from the basic spirit of the presentinvention. Accordingly, it will be appreciated by those skilled in theart that within the scope of the appended claims, the invention may bepracticed other than has been specifically described herein. Hence, theattached claims are intended to cover the invention embodied in theclaims and substantial equivalents thereto.

What is claimed is:
 1. An isolated microorganism of the strain BacillusATCC
 202137. 2. The microorganism of claim 1, determined to be speciesB. subtilis by the Fatty Acid Methyl Ester Analysis method usingTrypticase Soy Broth Agar version 3.9 at 28.0 C of the database ofMicrocheck, Inc. of Northfield, Vt.
 3. A combination comprising themicroorganism of claim 1 and one or more other microorganisms.
 4. Amethod of treating wastewater comprising the step of adding to thewastewater the combination of claim
 3. 5. A method of treating plumbingsystem components comprising the step of introducing into the componentsthe combination of claim
 3. 6. A method of treating animal feedcomprising the step of adding the combination of claim 4 to the feed. 7.A method of treating wastewater comprising the step of adding to thewastewater the microorganism of claim
 1. 8. A method of treatingplumbing system components comprising the step of introducing into thecomponents the microorganism of claim
 1. 9. A method of treating animalfeed comprising the step of adding the microorganism of claim 1 to thefeed.